ODELAY: um método em larga escala para a quantificação multiparamétrica do crescimento de fermento

The overall goal of ODELAY is to measure growth phenotypes of individual cells growing into colonies in a high throughput manner. This method can help answer key questions in microbiology, genetics, and systems biology such as the effects of genetic perturbations and drug interactions on growth phenotypes of any colony forming microorganism. The main advantage of ODELAY is that it enables the researcher to directly observe and quantify population heterogeneity amongst a large number of individuals.

Prior to assembling the agar mold clean the acrylic molds with 70%ethanol and dry them using forced air. There are three bottom pieces and two upright pieces. To begin assembly of the agar mold take the three bottom pieces and assemble them so that the two identical pieces sandwich the third piece.

Use two small binder clips to clamp the base pieces on each side. Place the uprights into the mold making sure that the laser curve is correctly positioned. Place a cleaned and dried glass slide on the mold and hold it in place while placing a second glass slide on the other side.

Clamp the assembly with a larger binder clip. Clamp down both slides with larger binder clips making sure each upper binder clip is in contact with the glass slide overlapping the acrylic. Before filling the assembled mold with molten agar ensure the edges are sealed by pipetting about 70 microliters of molten agar media along the inner side of the mold.

Fill the mold without trapping air bubbles inside by slowly pipetting the molten agar along the edge of the mold. After the mold is filled allow it to cool at about 23 degrees Celsius ambient temperature for 40-60 minutes. The next step is mold separation.

Start by removing the bottom binder clip. Then remove the bottom part of the mold that consists of the three sandwiched pieces. Hold the slides by compressing them and then remove the binder clips carefully.

Place the mold on the edge of the bench top so that the side of the mold with the blue dot is facing up. Place both thumbs underneath the acrylic and the first fingers on the top toward the inner edge of the mold and slowly and carefully push up with the thumbs against the mold as if pivoting the mold spacer about its lower edge. Apply constant but slowly increasing pressure.

A break and an air bubble should appear along a line where the top glass covers the mold spacer. After seeing the initial break-line continue pushing up with both thumbs and apply constant pressure. The agar should start to break away from the glass.

Continue to pivot the mold upwards. As the glass comes completely free grab it and remove it completely from the mold. Then remove the other mold piece using a similar motion to lift the piece free without moving the agar.

Place the slides in a sterile tip box with some sterile high purity water in the bottom. Close the box and store at 4 degrees Celsius overnight for use the following day. Start this procedure by preparing the strains in a 96 well plate as described in the text protocol.

Using a plate reader to measure the optical density at 600 nanometers of each culture. Next use an automated dilution protocol to dilute the cultures in the plate to an optical density at 600 nanometers of approximately 0.1. Once the dilution program is set up load plates, tubes, and tips onto the liquid handling robot's deck as indicated by the deck layout.

Click the play button to run the dilution program. Select the correct number of 50 microliter tips and the correct number of 300 microliter tips. When the dilution is done, remove the dilution plate from the robot and culture the plate at 30 degrees Celsius maintaining the humidity to prevent the plate drying out for five to six hours.

About one to two hours before the incubation of the culture plate is complete turn on the incubation chambers for the microscope and allow them to equilibrate at 30 degrees Celsius. Dilute the culture a second time to an optical density at 600 nanometers of 0.01 to 0.02 using the same automated dilution protocol. Cover the dilution plate with a metal freezer seal and sonicate in an ice bath for 30 seconds with the plate floating in ice water.

Label four 96 well flat bottom plates as one, two, three, and four. Transfer 150 microliters of each sonicated culture from the dilution plate to the flat bottom plates following this pattern. Wells A1 to D6 into wells C4 to F9 of Plate One.

Wells A7 to D12 into wells C4 to F9 of Plate Two. Wells E1 to H6 into wells C4 to F9 of Plate Three. And wells E7 to H12 into wells C4 to F9 of Plate Four.

To begin this procedure place the agarose slide correctly into the slide chamber. Next place the 96 well flat bottom plates on the table in order of their quadrant. Plates One, Two, Three and Four from left to right.

Lay out tips in four empty tip boxes so that the inner 24 wells of each tip box are occupied with tips. In a fifth box, place a tip in position C10 and tips with their end cut off in positions A1, A12, H1, and H12. These four cut off tips will provide stability for the tip plate clamp.

Place the first tip punch into the start site of the robot control spotting program to punch a hole in the agarose to be used later for aligning the origin coordinate. Place Plate One on the liquid handling robot to spot the first quadrant. Remove the lid and continue the spotting program.

Check to make sure all spots are present. Empty the used tips into the biohazard container And place fresh tips on the robot. Wait for about 30 seconds for the spots to dry to about one millimeter in diameter and then continue the program.

Swap out Plate One for Plate Two and continue the spotting program. When all four quadrants have been spotted and the spots are dry replace the slide chamber cover and flip the apparatus over. Place a glass slide on the top side of the slide chamber.

First load the slide chamber into the microscope. Time lapse microscopy is performed by running a custom designed script. Click on the shutter to open the transmitted light shutter and then the focus button to initiate the high camera rate.

Click on Go Origin"and move the stage to find the origin mark that was punched in the agar then move slightly to the right to focus on cells spotted in area E7.Move back to the origin punch and center it in the field of view. Set the origin to this value by pressing the Set"button. Move to position H18 and focus using the hex screw closest to that location.

Then move to position L7 and focus using the hex screw closest to that location. Finally move to position E7 and focus using the hex screw closest to that location. Check the focus in the center and at the edges at spots E12, H12, and L12 adjust the autofocus range if the focus z values as indicated by the z value are greater than plus or minus the autofocus range.

Press the Reset"button and then press ODELAY. Choose the directory to save the data. The microscope will collect data for 48 hours or until the program is closed.

These representative timelapse images show yeast cells growing on solid medium as zero hour, three hours, six hours, and nine hours after spotting. Shown here is a representative data set comparing two yeast strains after processing the timelapse images. The relatively uniform doubling times reflect the well-prepared agarose slide, however the lag times vary considerably due to autofocus settings that are not optimal.

A more consistent data set is shown here in which all doubling times appear to overlap well and the lag times appear to be consistent. Once mastered ODELAY's setup can be done in three to four hours if it is performed properly. The most critical steps for repeatable ODELAY measurements are preparing the media consistently and avoiding mechanically deforming the media while separating the slides.

ODELAY is a very sensitive assay. For example it can observe growth defects in temperature sensitive yeast strains at room temperature where commonly used spot based assays require culturing yeast at 37 degrees Centigrade to reveal a growth defect. While ODELAY has been developed in yeast, the method is applicable to any colony-forming organism including pathogens to explore a wide range of environmental and genetic conditions to understand the behavior of microorganisms.

After watching this video you should have a good understanding of how to perform ODELAY to measure growth phenotypes of single celled microorganisms forming colonies on solid media. Don't forget that working with microorganisms can be hazardous and precautions such as wearing personal protective equipment suitable for the organisms studied should always be taken.